To fully understand learning and the formation of memory, it is necessary to understand both the basic mechanisms responsible for the induction and consolidation of memory, as well as the processes responsible for the modulation of those basic processes. Many factors modulate the processes of memory formation including general health, stress, motivation, age and the time of day. The long-term objectives of our research are to understand the modulation of long-term associative memory formation by the circadian clock including the underlying molecular mechanisms, the physiological function and the behavioral consequences. The circadian clock regulates long-term memory formation in Aplysia californica, such that animals form robust long-term memory when trained during the day, but no long-term memory when trained at night. We will investigate the signaling pathways involved in an operant, associative form of learning in Aplysia, learning that food is inedible (LFI), and modulation by the circadian clock using behavioral, pharmacological and biochemical assays. In Specific Aim 1, we will identify the kinase signaling pathways necessary for LFI and determine whether the activation of these kinases is modulated by the circadian clock. Specifically, we will determine whether PKG, MARK and PKA signaling are necessary for LFI. The goal of Aim 2 is to determine whether the circadian clock modulates learning-induced transcription for LFI memory and whether the transcription factor ApC/EBP is involved in LFI. Specific Aim 3 examines the effect, at the molecular level, of training animals at night when they only form short-term memories and investigates methods of converting the partial memory into long-term memory. In Aim 4, we examine negative regulatory elements in long-term memory formation. We will investigate whether the circadian clock regulates p38 kinase activity or phosphatase activity. This research will significantly contribute to our understanding of the molecular mechanisms underlying associative operant learning in Aplysia as well as greatly furthering our understanding of the regulation of output behaviors by the circadian clock. One objective of the proposed experiments is to determine how circadian suppression of long-term memory formation at night may be relieved to improve memory formation at night. Thus, this research will provide a basis of research for future therapeutic treatments to improve memory and performance.